Apparatus, system, and method for collecting a target material

ABSTRACT

This disclosure is directed to an apparatus, system and method for retrieving a target material from a suspension. A system includes a processing vessel, such as an Eppendorf tube, a syringe or a test tube, and a collector. The collector is sized and shaped to fit into a primary vessel, such as a test tube. The collector funnels the target material from the suspension through a cannula and into the processing vessel. The cannula extends into a cavity at a first end of the collector that holds the processing vessel. The collector includes a funnel at a second end in fluid communication with the cannula. In one implementation, the processing vessel includes at least one displacement fluid to be expelled, such that the at least one displacement fluid pushes the target material into the collector.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a continuation of application Ser. No. 14/495,449,which is a continuation-in-part of application Ser. No. 14/090,337,which claims the benefit of Provisional Application No. 61/732,029,filed Nov. 30, 2012, Provisional Application No. 61/745,094, filed Dec.21, 2012, Provisional Application No. 61/791,883, filed Mar. 15, 2013,Provisional Application No. 61/818,301, filed May 1, 2013, andProvisional Application No. 61/869,866, filed Aug. 26, 2013.

TECHNICAL FIELD

This disclosure relates generally to density-based fluid separation and,in particular, to retrieving a target material from a suspension.

BACKGROUND

Suspensions often include materials of interests that are difficult todetect, extract and isolate for analysis. For instance, whole blood is asuspension of materials in a fluid. The materials include billions ofred and white blood cells and platelets in a proteinaceous fluid calledplasma. Whole blood is routinely examined for the presence of abnormalorganisms or cells, such as ova, fetal cells, endothelial cells,parasites, bacteria, and inflammatory cells, and viruses, including HIV,cytomegalovirus, hepatitis C virus, and Epstein-Barr virus. Currently,practitioners, researchers, and those working with blood samples try toseparate, isolate, and extract certain components of a peripheral bloodsample for examination. Typical techniques used to analyze a bloodsample include the steps of smearing a film of blood on a slide andstaining the film in a way that enables certain components to beexamined by bright field or fluorescence microscopy.

On the other hand, materials of interest that occur in a suspension withvery low concentrations are especially difficult if not impossible todetect and analyze using many existing techniques. Consider, forinstance, circulating tumor cells (“CTCs”), which are cancer cells thathave detached from a tumor, circulate in the bloodstream, and may beregarded as seeds for subsequent growth of additional tumors (i.e.,metastasis) in different tissues. The ability to accurately detect andanalyze CTCs is of particular interest to oncologists and cancerresearchers. However, CTCs occur in very low numbers in peripheral wholeblood samples. For instance, a 7.5 ml sample of peripheral whole bloodsample that contains as few as 5 CTCs is considered clinically relevantfor the diagnosis and treatment of a cancer patient. In other words,detecting 5 CTCs in a 7.5 ml blood sample is equivalent to detecting 1CTC in a background of about 10 billion red and white blood cells, whichis extremely time consuming, costly and difficult to accomplish usingblood film analysis.

As a result, practitioners, researchers, and those working withsuspensions continue to seek systems and methods for accurate analysisof suspensions for the presence or absence rare materials of interest.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B show an example collector.

FIGS. 2A-2B show an example collector.

FIGS. 2C-2D show an example collector.

FIGS. 3A-3B show an example collector-processing vessel system.

FIGS. 4A-4B show an example collector-canopy system.

FIGS. 5A-5B show an example sealing ring.

FIGS. 5C-5D show an example sealing ring.

FIGS. 5E-5F show an example sealing ring.

FIG. 5G shows an example sealing ring.

FIG. 6 shows a flow diagram of an example method for retrieving a targetmaterial.

FIGS. 7A-7B show example float and primary vessel systems.

FIG. 8 shows an example float and primary vessel system having undergonedensity-based separation.

FIG. 9 shows an example sealing ring and the example float and primaryvessel system forming a seal.

FIGS. 10A-10G show an example system retrieving a target material.

DETAILED DESCRIPTION

This disclosure is directed to an apparatus, system and method forretrieving a target material from a suspension. A system includes aprocessing vessel, such as an Eppendorf tube, a syringe or a test tube,and a collector. The collector is sized and shaped to fit into a primaryvessel, such as a test tube. The collector funnels the target materialfrom the suspension through a cannula and into the processing vessel.The cannula extends into a cavity at a first end of the collector thatholds the processing vessel. The collector includes a funnel at a secondend in fluid communication with the cannula. In one implementation, theprocessing vessel includes at least one displacement fluid to beexpelled, such that the at least one displacement fluid pushes thetarget material into the collector.

Collector

FIG. 1A shows an isometric view of a collector 100. FIG. 1B shows across-sectional view of the collector 100 taken along the line I-I shownin FIG. 1A. Dot-dashed line 102 represents the central orhighest-symmetry axis of the collector 100. The collector 100 may besized and shaped to fit within a primary vessel containing or capable ofholding a suspension, the suspension suspected of including a targetmaterial. The collector 100 funnels the target material from thesuspension through a cannula 106 and into a processing vessel (notshown) to be located within a cavity 108. The collector 100 includes themain body 104 which includes a first end 110 and a second end 112. Aseal may be formed between the second end 112 and an inner wall of theprimary vessel to maintain a fluid-tight sealing engagement before,during, and after centrifugation and to inhibit any portion of thesuspension from being located or flowing between an inner wall of theprimary vessel and a main body 104 of the collector 100. The seal may beformed by an interference fit, a grease (such as vacuum grease), anadhesive, an epoxy, by bonding (such as by thermal bonding), by welding(such as by ultrasonic welding), by clamping (such as with a ring orclamp), an insert (such as an O-ring or a collar) that fits between thesecond end 112 and the inner wall of the primary vessel, or the like.The main body 104 may be any appropriate shape, including, but notlimited to, cylindrical, triangular, square, rectangular, or the like.The collector 100 also includes an internal funnel 114 which is aconcave opening. The funnel 114 may taper toward the cannula 106 fromthe second end 112. The funnel 114 channels a target material from belowthe second end 112 into the cannula 106 which is connected to, and influid communication with, an apex of the funnel 114. The apex of thefunnel 114 has a smaller diameter than the mouth of the funnel 114. Thefunnel 114 is formed by a tapered wall that may be straight,curvilinear, arcuate, or the like. The funnel 114 may be any appropriateshape, including, but not limited to, tubular, spherical, domed,conical, rectangular, pyramidal, or the like. Furthermore, the outermostdiameter or edge of the funnel 114 may be in continuous communication orconstant contact (i.e. sit flush) with the inner wall of the primaryvessel such that no dead space is present between the second end 112 ofthe collector 100 and the inner wall of the primary vessel.

The cannula 106, such as a tube or a needle, including, but not limitedto a non-coring needle, extends from the apex of the funnel 114 and intothe cavity 108. In the example of FIG. 1, the cavity 108 is a concaveopening extending from the first end 110 into the main body 104 and mayaccept and support the processing vessel (not shown). The cavity 108 maybe any appropriate depth to accept and support the processing vessel(not shown). The cannula 106 may extend any appropriate distance intothe cavity 108 in order to puncture the base of, or be inserted into,the processing vessel (not shown). The cannula 106 may include a flattip, a beveled tip, a sharpened tip, or a tapered tip. Furthermore, thecavity 108 may be any appropriate shape, including, but not limited to,tubular, spherical, domed, conical, rectangular, pyramidal, or the like.The cavity 108 may be threaded to engage a threaded portion of theprocessing vessel (not shown).

The collector 100 may also include a retainer (not shown) to prevent thecollector 100 from sliding relative to the primary vessel, therebykeeping the collector 100 at a pre-determined height within the primaryvessel. The retainer (not shown) may be a shoulder extending radiallyfrom the first end 110, a clip, a circular protrusion that extendsbeyond the circumference of the cylindrical main body 104, a detent, orthe like.

FIG. 2A shows an isometric view of a collector 200. FIG. 2B shows across-section view of the collector 200 taken along the line II-II shownin FIG. 2A. Dot-dashed line 202 represents the central orhighest-symmetry axis of the collector 200. The collector 200 is similarto the collector 100, except that the collector 200 includes a main body204 that is more elongated than the main body of the collector 100 inorder to accommodate a greater portion of the processing vessel (notshown). The main body 204 includes a first end 206 and a second end 208.A seal may be formed between the second end 208 and an inner wall of theprimary vessel to maintain a fluid-tight sealing engagement before,during, and after centrifugation and to inhibit any portion of thesuspension flowing between an inner wall of the primary vessel and themain body 204 of the collector 200. The seal may be formed by aninterference fit, a grease (such as vacuum grease), an adhesive, anepoxy, by bonding (such as thermal bonding), by welding (such asultrasonic welding), clamping (such as with a ring or clamp), an insert(such as an O-ring or a collar) that fits between the second end 208 andthe inner wall of the primary vessel, or the like.

The first end 206 includes a cavity 212 dimensioned to accept and holdat least a portion of the processing vessel (not shown). The cavity 212may have a tapered or stepped bottom end 220 on which the processingvessel (not shown) may rest. The first end 206 may also include at leastone cut-out 210 to permit proper grip of the processing vessel (notshown) for insertion and removal. The collector 200 funnels the targetmaterial from the suspension into an internal funnel 222 at the secondend 208, through a cannula 214, and into a processing vessel (not shown)located within the cavity 212. The cannula 214 may rest on a shelf 224so that an inner bore of the cannula 214 sits flush with an inner wallof the funnel 222, as shown in FIG. 2B.

The collector 200 may include a shoulder 216, which extendscircumferentially around the main body 204. The shoulder 216 may belarger than the inner diameter of the primary vessel so as to rest onthe open end of the primary vessel and, upon applying a lock ring (notshown) to the outside of the primary vessel and the shoulder 216, toinhibit movement of the collector 200 relative to the primary vessel.The lock ring (not shown) applies pressure to the primary vessel alongthe shoulder 216. The lock ring may be a two-piece ring, a one piecering wrapping around the full circumference of the primary vessel, or aone piece ring wrapping around less than the full circumference of theprimary vessel, such as one-half (½), five-eighths (⅝), two-thirds (⅔),three-quarters (¾), seven-eighths (⅞), or the like. Alternatively, theshoulder 216 may fit within the primary vessel. Alternatively, theshoulder 216 may be a clip, such that the shoulder 216 may include acatch into which the primary vessel may be inserted to inhibit movementof the collector 200 relative to the primary vessel. Alternatively, theshoulder 216 may form an interference fit with the inner wall of theprimary vessel around which a seal ring may be placed.

As shown in FIG. 2A, the collector 200 may include at least one window218 to access the cavity 212 through an inner wall of the main body 204.The at least one window 218 permits an operator to confirm properplacement of the processing vessel (not shown) within the cavity 212.The at least one window 218 also allows fluid discharged from thecannula 214 to flow out of the collector 200 and into a space formedbetween the collector 200 and the primary vessel (not shown) and abovethe seal between the second end 208 and the inner wall of the primaryvessel.

FIG. 2C shows an isometric view of a collector 230. FIG. 2D shows across-section view of the collector 230 taken along the line III-IIIshown in FIG. 2C. The collector 230 is similar to the collector 200,except that the collector 230 includes a main body 238 including anextension 234 extending away from a first end 232 and a lid 236 to atleast temporarily seal an opening 240 within the extension 234. Theopening 240 may be in fluid communication with the cavity 212 at thefirst end 232. The lid 236 may removable, puncturable and resealable(e.g. a flap lid), or puncturable and non-resealable (e.g. a foil lid).The extension 234 may be sized to accept the lid 236 when punctured suchthat a portion of the lid 236 does not extend into the cavity 212 at thefirst end 232. Note that the collector 230 does not include the at leastone cut-out 210.

The main body can be composed of a variety of different materialsincluding, but not limited to, a ceramic; metals; organic or inorganicmaterials; and plastic materials, such as polyoxymethylene (“Delrin®”),polystyrene, acrylonitrile butadiene styrene (“ABS”) copolymers,aromatic polycarbonates, aromatic polyesters, carboxymethylcellulose,ethyl cellulose, ethylene vinyl acetate copolymers, nylon, polyacetals,polyacetates, polyacrylonitrile and other nitrile resins,polyacrylonitrile-vinyl chloride copolymer, polyamides, aromaticpolyamides (“aramids”), polyamide-imide, polyarylates, polyaryleneoxides, polyarylene sulfides, polyarylsulfones, polybenzimidazole,polybutylene terephthalate, polycarbonates, polyester, polyester imides,polyether sulfones, polyetherimides, polyetherketones,polyetheretherketones, polyethylene terephthalate, polyimides,polymethacrylate, polyolefins (e.g., polyethylene, polypropylene),polyallomers, polyoxadiazole, polyparaxylene, polyphenylene oxides(PPO), modified PPOs, polystyrene, polysulfone, fluorine containingpolymer such as polytetrafluoroethylene, polyurethane, polyvinylacetate, polyvinyl alcohol, polyvinyl halides such as polyvinylchloride, polyvinyl chloride-vinyl acetate copolymer, polyvinylpyrrolidone, polyvinylidene chloride, specialty polymers, polystyrene,polycarbonate, polypropylene, acrylonitrite butadiene-styrene copolymer,butyl rubber, ethylene propylene diene monomer; and combinationsthereof.

The cannula can be composed of a variety of different materialsincluding, but not limited to, a ceramic; metals; organic or inorganicmaterials; and plastic materials, such as a polypropylene, acrylic,polycarbonate, or the like; and combinations thereof. The cannula mayhave a tip along a longitudinal axis of the cannula.

Collector-Processing Vessel System

FIG. 3A shows an exploded view of the example collector 200 andprocessing vessel 302. FIG. 3B shows a cross-sectional view of theprocessing vessel 302 inserted into the cavity 212 at the first end 206of the collector 200 taken along the line IV-IV shown in FIG. 3A. Thecollector 200 and processing vessel 302 form a collector-processingvessel system 300. The processing vessel 302 may be an Eppendorf tube, asyringe, or a test tube and has a closed end 304 and an open end 306.The open end 306 is sized to receive a cap 308. The cap 308 may becomposed of re-sealable rubber or other suitable re-sealable materialthat can be repeatedly punctured with a needle or other sharp implementto access the contents stored in the processing vessel 302 interior andre-seals when the needle or implement is removed. Alternatively, theprocessing vessel 302 may also have two open ends that are sized toreceive caps. The processing vessel 302 may have a tapered geometry thatwidens or narrows toward the open end 306; the processing vessel 302 mayhave a generally cylindrical geometry; or, the processing vessel 302 mayhave a generally cylindrical geometry in a first segment and acone-shaped geometry in a second segment, where the first and secondsegments are connected and continuous with each other. Although at leastone segment of the processing vessel 302 has a circular cross-section,in other embodiments, the at least one segment can have elliptical,square, triangular, rectangular, octagonal, or any other suitablecross-sectional shape. The processing vessel 302 can be composed of atransparent, semitransparent, opaque, or translucent material, such asplastic or another suitable material. The processing vessel includes acentral axis 314, which when inserted into the cavity 212 is coaxialwith the central axis 202 of the collector 200. The processing vessel302 may also include a plug 310 at the closed end 304 to permit theintroduction of the target material or to exchange the target materialwith a displacement fluid 312. The closed end 304 may be threaded toprovide for a threaded connection with a threaded cavity 212 of thecollector 200. The processing vessel 302 may be composed of glass,plastic, or other suitable material.

The plug 310 may be composed of re-sealable rubber or other suitablere-sealable material that can be repeatedly punctured with a needle orother sharp implement to access the contents of the processing vessel302 interior or permit introduction of contents into the processingvessel 302 and re-seals when the needle or implement is removed. Theplug 310 may be inserted into the processing vessel 302 such that a sealis maintained between the plug 310 and the processing vessel 302, suchas by an interference fit. Alternatively, the plug 310 can be formed inthe closed end 304 of the processing vessel 302 using heated liquidrubber that can be shaped while warm or hot and hardens as the rubbercools. An adhesive may be used to attach a plug 310 to the inner wall ofthe processing vessel can be a polymer-based adhesive, an epoxy, acontact adhesive or any other suitable material for bonding or creatinga thermal bond. Alternatively, the plug 310 may be injected into theprocessing vessel 302. Alternatively, the plug 310 may be thermallybonded to the processing vessel 302.

In the example of FIG. 3B, the cannula 214 has a tapered tip thatpunctures the plug 310 and extends into an inner cavity of theprocessing vessel 302 with the shaft of the cannula 214 not extendinginto the inner cavity of the processing vessel 302. As explained ingreater detail below, the inner cavity of the processing vessel 302holds the target material. The cannula 214 may be covered by aresealable sleeve (not shown) to prevent the target material fromflowing out unless the processing vessel 302 is in the cavity 212 to adepth that allows the cannula 214 to just penetrate the processingvessel 302. The resealable sleeve (not shown) covers the cannula 214, isspring-resilient, can be penetrated by the cannula 214, and is made ofan elastomeric material capable of withstanding repeated punctures whilestill maintaining a seal.

As shown in FIGS. 3A-3B, the processing vessel 302 may be loaded with adisplacement fluid 312 prior to insertion into the collector 200. Thedisplacement fluid 312 displaces the target material, such that when thecollector 200 and processing vessel 302 are inserted into the primaryvessel (not shown) including the target material, and the collector,processing vessel, and primary vessel undergo centrifugation, thedisplacement fluid 312 flows out of the processing vessel 302 and intothe primary vessel, and, through displacement, such as through buoyantdisplacement (i.e. lifting a material upwards), pushes the targetmaterial through the cannula 214 and into the processing vessel 302.

The displacement fluid 312 has a greater density than the density of thetarget material of the suspension (the density may be greater than thedensity of a subset of suspension fractions or all of the suspensionfractions) and is inert with respect to the suspension materials. Thedisplacement fluid 312 may be miscible or immiscible in the suspensionfluid. Examples of suitable displacement fluids include, but are notlimited to, solution of colloidal silica particles coated withpolyvinylpyrrolidone (e.g. Percoll), polysaccharide solution (e.g.Ficoll), iodixanol (e.g. OptiPrep), an organic solvent, a liquid wax, anoil, a gas, and combinations thereof; olive oil, mineral oil, siliconeoil, immersion oil, mineral oil, paraffin oil, silicon oil,fluorosilicone, perfluorodecalin, perfluoroperhydrophenanthrene,perfluorooctylbromide, and combinations thereof; organic solvents suchas 1,4-Dioxane, acetonitrile, ethyl acetate, tert-butanol,cyclohexanone, methylene chloride, tert-Amyl alcohol, tert-Butyl methylether, butyl acetate, hexanol, nitrobenzene, toluene, octanol, octane,propylene carbonate, tetramethylene sulfones, and ionic liquids;polymer-based solutions; surfactants; perfluoroketones, such asperfluorocyclopentanone and perfluorocyclohexanone, fluorinated ketones,hydrofluoroethers, hydrofluorocarbons, perfluorocarbons,perfluoropolyethers, silicon and silicon-based liquids, such asphenylmethyl siloxane; and combinations thereof.

The processing vessel 302 may also include a processing solution (notshown) to effect a transformation on the target material when the targetmaterial enters the processing vessel 302. The processing solution (notshown) may be a preservative, a cell adhesion solution, a dye, or thelike. Unlike the displacement fluid 312, most, if not all, of theprocessing solution (not shown) remains within the processing vessel 302upon centrifugation, thereby effecting the transformation on the targetmaterial in one manner or another (i.e. preserving, increasing adhesionproperties, or the like). The processing solution (not shown) may beintroduced as a liquid or as a liquid contained in a casing. The casingmay be dissolvable in an aqueous solution but not in the displacementfluid 312 (such as gel cap); or, the casing may be breakable, such thatthe casing breaks when the processing vessel 302 is shaken in a vortexmixer. Additionally, more than one processing solution may be used.

The processing vessel 302 may include a flexible cap that can be pushedto dispense a pre-determined volume therefrom and onto a substrate, suchas a slide or a well plate. The cap 308 may be flexible or the cap 308may be removed and the flexible cap inserted into the open end 306.Alternatively, the processing vessel 302 may be attached to (i.e. afteraccumulating the target material) or may include a dispenser, which iscapable of dispensing a pre-determined volume of target material fromthe processing vessel 302 onto another substrate, such as a microscopeslide. The dispenser may repeatedly puncture the re-sealable cap 308 orcompress the material within the processing vessel 302 to withdraw anddispense the pre-determined volume of target material onto thesubstrate. Alternatively, the cap 308 may be removed and the dispenser(not shown) may be inserted directly into the processing vessel 302 todispense the buffy coat-processing solution mixture.

Collector-Canopy System

FIG. 4A shows an exploded view of the example collector 200 and canopy402. FIG. 4B shows a cross-sectional view of the processing vessel 402inserted into the cavity 212 of the collector 200 taken along the lineV-V shown in FIG. 4A. The collector 200 and canopy 402 form acollector-canopy system 400. The canopy 402 is similar to the processingvessel 302, except that the canopy has a second open end 404. When thecollector-canopy system 400 is inserted into the primary vessel, somefluid within the primary vessel, such as a portion of the suspension, aportion of a suspension fraction, a portion of a clearing fluid, or thelike, may be discharged through the cannula 214. The canopy 402 inhibitsa portion of the fluid in the primary vessel that may be dischargedthrough the cannula 214 from escaping from the opening of the first end206 of the collector 200. The discharged fluid, having been blocked bythe canopy 402, flows out of the second open end 404, and out of thewindow 218. Dashed lines 406 show fluid flow as the fluid is dischargedthrough the cannula 214 and retained by the canopy 402.

Alternatively, when the collector 230 is used, the lid 236 of thecollector 230 inhibits a portion of the fluid in the primary vessel thatmay be discharged through the cannula 214 from escaping from the openingof the first end 206 of the collector 200 in a manner similar to that ofthe canopy 402.

Sealing Ring

FIG. 5A shows an isometric view of a sealing ring 500. FIG. 5B shows atop down view of the sealing ring 500. Dot-dashed line 502 representsthe central or highest-symmetry axis of the sealing ring 500. Thesealing ring 500 includes an inner wall 504, an outer wall 506, and acavity 508. In FIG. 5B, R_(IW) represents the radial distance from thecenter of the sealing ring 500 to the inner wall 504, and R_(OW)represents the radial distance from the center of the sealing ring 500to the outer wall 506. The sealing ring 500 is configured to fit arounda primary vessel, such as a tube. The cavity 508 is sized and shaped toreceive the primary vessel. The sealing ring 500 may be tightened, suchthat the size of the cavity 508 and the radii of the inner and outerwalls 504 and 506 are reduced by circumferentially applying anapproximately uniform, radial force, such as the radial force created bya clamp, around the outer wall 506 directed to the central axis 502 ofthe sealing ring 500. When the sealing ring 500 is tightened around theprimary vessel, the uniform force applied to the sealing ring 500 isapplied to the primary vessel, thereby causing the primary vessel toconstrict. When the radial force is removed from the sealing ring 500,the sealing ring 500 remains tightened and in tension around the primaryvessel.

The sealing ring may be any shape, including, but not limited to,circular, triangular, or polyhedral. FIG. 5C shows an isometric view ofa sealing ring 510. FIG. 5D shows a top down view of the sealing ring510. Sealing ring 510 is similar to sealing ring 500, except sealingring 510 is polyhedral. Dot-dashed line 512 represents the central orhighest-symmetry axis of the sealing ring 510. The sealing ring 510includes an inner wall 514, an outer wall 516, and a cavity 518. Thesealing ring may be composed of a metal, such as brass, a polymer, orcombinations thereof.

Alternatively, as shown in FIG. 5E, a sealing ring 520 may be composedof a piezoelectric material. FIG. 5F shows a top down view of thesealing ring 520. Dot-dashed line 522 represents the central orhighest-symmetry axis of the sealing ring 520. The sealing ring 520 maybe connected to an electric potential source 528, such as a battery, viaa first lead 524 and a second lead 526. The electric potential source528 creates a mechanical strain that causes the sealing ring 520 totighten (i.e. sealing ring 520 radii decrease). The sealing ring 520includes an inner wall 530, an outer wall 532, and a cavity 534. In FIG.5F, R_(IW) represents the radial distance from the center of the sealingring 520 to the inner wall 530, and R_(OW) represents the radialdistance from the center of the sealing ring 520 to the outer wall 532.Alternatively, the sealing ring 520 may be in a naturally tightenedstated. When applying the electric potential the sealing ring 520expands. Alternatively, a portion of the sealing ring may be composed ofthe piezoelectric material, such that the piezoelectric portion acts asan actuator to cause the other portion of the sealing ring to tightenand apply the substantially uniform circumferential pressure on theprimary vessel, thereby constricting the primary vessel to form theseal.

FIG. 5G shows an isometric view of a sealing ring 540. The sealing ringincludes an adjustment mechanism 548 to adjust the inner diameterR_(ID). The collapsible ring includes a first end 542 and a second end546, the first and second ends 542 and 546 being joined by a bandportion 544. The first and second ends 542 and 546 include complementaryportions of the adjustment mechanism 548. The adjustment mechanism 548includes, but is not limited to, a ratchet, tongue and groove, detents,or the like.

The sealing ring may also include a thermal element, such as a heatedwire. The thermal element may soften the primary vessel forconstriction. Alternatively, the thermal element may melt the primaryvessel to provide a more adherent seal. Alternatively, the thermalelement may cause the sealing ring to compress, thereby forming a sealbetween the primary vessel and float.

Method

For the sake of convenience, the methods are described with reference toan example suspension of anticoagulated whole blood. But the methodsdescribed below are not intended to be so limited in their scope ofapplication. The methods, in practice, can be used with any kind ofsuspension. For example, a sample suspension can be urine, blood, bonemarrow, cystic fluid, ascites fluid, stool, semen, cerebrospinal fluid,nipple aspirate fluid, saliva, amniotic fluid, vaginal secretions, mucusmembrane secretions, aqueous humor, vitreous humor, vomit, and any otherphysiological fluid or semi-solid. It should also be understood that atarget material can be a fraction of a sample suspension, such as buffycoat, a cell, such as ova, a fetal cell, a fetal nucleated red bloodcell, or a circulating tumor cell (“CTC”), a circulating endothelialcell, a fetal cell, a vesicle, a liposome, a protein, a nucleic acid, abiological molecule, a naturally occurring or artificially preparedmicroscopic unit having an enclosed membrane, parasites, microorganisms,viruses, or inflammatory cells.

FIG. 6 shows a flow diagram for an example method for retrieving atarget material. In block 602, a suspension, such as anticoagulatedwhole blood, is obtained. In block 604, the whole blood is added to aprimary vessel, such as a test tube. A float may also be added to theprimary vessel. For the sake of convenience, the methods are describedwith reference to the float, but the methods described below are notintended to be so limited in their application and may be performedwithout the float.

FIG. 7A shows an isometric view of an example primary vessel and floatsystem 700. The system 700 includes a primary vessel 702 and a float 704suspended within whole blood 706. In the example of FIG. 7A, the primaryvessel 702 has a circular cross-section, a first open end 710, and asecond closed end 708. The open end 710 is sized to receive a cap 712.The primary vessel may also have two open ends that are sized to receivecaps, such as the example tube and separable float system 720 shown FIG.7B. The system 720 is similar to the system 700 except the primaryvessel 702 is replaced by a primary vessel 722 that includes two openends 724 and 726 configured to receive the cap 712 and a cap 728,respectively. The primary vessels 702 and 722 have a generallycylindrical geometry, but may also have a tapered geometry that widens,narrows, or a combination thereof toward the open ends 710 and 724,respectively. Although the primary vessels 702 and 722 have a circularcross-section, in other embodiments, the primary vessels 702 and 722 canhave elliptical, square, triangular, rectangular, octagonal, or anyother suitable cross-sectional shape that substantially extends thelength of the tube. The primary vessels 702 and 722 can be composed of atransparent, semitransparent, opaque, or translucent material, such asplastic or another suitable material. The primary vessels 702 and 722each include a central axis 718 and 730, respectively. The primaryvessel 702 may also include a septum 714, as seen in magnified view 716,at the closed end 708 to permit the removal of a fluid, the suspension,or a suspension fraction, whether with a syringe, a pump, by draining,or the like. The primary vessel 702 may have an inner wall and a firstdiameter.

The septum 714 may be composed of re-sealable rubber or other suitablere-sealable material that can be repeatedly punctured with a needle orother sharp implement to access the contents of the primary vessel 702interior and re-seals when the needle or implement is removed. Theseptum 714 may be inserted into the primary vessel 702 such that a sealis maintained between the septum 714 and the primary vessel 702, such asby an interference fit. Alternatively, the septum 714 can be formed inthe openings and/or the bottom interior of the tube using heated liquidrubber that can be shaped while warm or hot and hardens as the rubbercools. An adhesive may be used to attach the septum 714 to the wall ofthe opening and tube interior and can be a polymer-based adhesive, anepoxy, a contact adhesive or any other suitable material for bondingrubber to plastic or creating a thermal bond. Alternatively, the septum714 may be thermally bonded to the primary vessel 702.

The float 704 includes a main body, two teardrop-shaped end caps, andsupport members radially spaced and axially oriented on the main body.Alternatively, the float 704 may not include any support members.Alternatively, the float 704 may include support members which do notengage the inner wall of the primary vessel 702.

In alternative embodiments, the number of support members, supportmember spacing, and support member thickness can each be independentlyvaried. The support members can also be broken or segmented. The mainbody is sized to have an outer diameter that is less than the innerdiameter of the primary vessel 702, thereby defining fluid retentionchannels between the outer surface of the main body and the inner wallof the primary vessel 702. The surfaces of the main body between thesupport members can be flat, curved or have another suitable geometry.The support members and the main body may be a singular structure or maybe separate structures.

Embodiments include other types of geometric shapes for float end caps.The top end cap may be teardrop-shaped, dome-shaped, cone-shaped, or anyother appropriate shape. The bottom end cap may be teardrop-shaped,dome-shaped, cone-shaped, or any other appropriate shape. In otherembodiments, the main body of the float 704 can include a variety ofdifferent support structures for separating samples, supporting the tubewall, or directing the suspension fluid around the float duringcentrifugation. Embodiments are not intended to be limited to theseexamples. The main body may include a number of protrusions that providesupport for the tube. In alternative embodiments, the number and patternof protrusions can be varied. The main body may include a singlecontinuous helical structure or shoulder that spirals around the mainbody creating a helical channel. In other embodiments, the helicalshoulder can be rounded or broken or segmented to allow fluid to flowbetween adjacent turns of the helical shoulder. In various embodiments,the helical shoulder spacing and rib thickness can be independentlyvaried. In another embodiment, the main body may include a supportmember extending radially from and circumferentially around the mainbody. In another embodiment, the support members may be tapered.

The float 704 can be composed of a variety of different materialsincluding, but not limited to, metals; organic or inorganic materials;ferrous plastics; sintered metal; machined metal; plastic materials andcombinations thereof. The primary vessel 702 may have an inner wall anda first diameter. The float 704 can be captured within the primaryvessel 702 by an interference fit, such that under centrifugation, aninner wall of the tube expands to permit axial movement of the float704. When centrifugation stops, the inner wall reduces back to the firstdiameter to induce the interference fit. Alternatively, the inner wallmay not expand and the interference fit may not occur between the float704 and the primary vessel 702, such that the float moves freely withinthe tube before, during, or after centrifugation. The end caps of thefloat may be manufactured as a portion of the main body, thereby beingone singular structure, by machining, injection molding, additivetechniques, or the like; or, the end caps may be connected to the mainbody by a press fit, an adhesive, a screw, any other appropriate methodby which to hold at least two pieces together, or combinations thereof.

The cap 712 may be composed of a variety of different materialsincluding, but not limited to, organic or inorganic materials; plasticmaterials; and combination thereof.

Returning to FIG. 6, in block 606, the primary vessel, the float, andthe whole blood undergo density-based separation, such as bycentrifugation, thereby permitting separation of the whole blood intodensity-based fractions along an axial position in the tube based ondensity. FIG. 8 shows an isometric view of the primary vessel and floatsystem 700 having undergone density-based separation, such as bycentrifugation. Suppose, for example, the centrifuged whole bloodincludes three fractions. For convenience sake, the three fractionsinclude plasma, buffy coat, and red blood cells. However, when anothersuspension undergoes centrifugation, there may be more than, less than,or the same number of fractions, each fraction having a differentdensity. The suspension undergoes axial separation into three fractionsalong the length the tube based on density, with red blood cells 803located on the bottom, plasma 801 located on top, and buffy coat 802located in between, as shown in FIG. 8. The float 704 may have anyappropriate density to settle within one of the fractions. The densityof the float 704 can be selected so that the float 704 expands the buffycoat 802 between the main body of the float and the inner wall of theprimary vessel. The buffy coat 802 can be trapped within an area betweenthe float 704 and the primary vessel 702.

At least one delineation fluid (not shown) may be used to providefurther separation between the target material and any non-targetmaterial above and/or below the target material. The at least onedelineation fluid (not shown) may have a density greater than or lessthan the target material. For example, when it is desirous to furtherseparate the buffy coat 802 and the red blood cells 803, the delineationfluid may have a density greater than the buffy coat 802 and less thanthe red blood cells 803. The at least one delineation fluid (not shown)may be miscible or immiscible with the suspension fluid and inert withrespect to the suspension materials. The at least one delineation fluid(not shown) may also provide an area in which to seal the primary vessel702, because there is greater delineation and separation between thebuffy coat 802 and the red blood cells 803. The at least one delineationfluid (not shown) may be used whether or not a float is used. Examplesof suitable delineation fluids include, but are not limited to, solutionof colloidal silica particles coated with polyvinylpyrrolidone (e.g.Percoll), polysaccharide solution (e.g. Ficoll), iodixanol (e.g.OptiPrep), cesium chloride, sucrose, sugar-based solutions,polymer-based solutions, surfactants, an organic solvent, a liquid wax,an oil, a gas, and combinations thereof; olive oil, mineral oil,silicone oil, immersion oil, mineral oil, paraffin oil, silicon oil,fluorosilicone, perfluorodecalin, perfluoroperhydrophenanthrene,perfluorooctylbromide, and combinations thereof; organic solvents suchas 1,4-Dioxane, acetonitrile, ethyl acetate, tert-butanol,cyclohexanone, methylene chloride, tert-Amyl alcohol, tert-Butyl methylether, butyl acetate, hexanol, nitrobenzene, toluene, octanol, octane,propylene carbonate, tetramethylene sulfones, and ionic liquids;polymer-based solutions; surfactants; perfluoroketones, such asperfluorocyclopentanone and perfluorocyclohexanone, fluorinated ketones,hydrofluoroethers, hydrofluorocarbons, perfluorocarbons,perfluoropolyethers, silicon and silicon-based liquids, such asphenylmethyl siloxane; and combinations thereof.

FIG. 9 shows a seal being formed to prevent fluids from moving up ordown within the primary vessel. The seal also inhibits float movement.The sealing ring 500 exerts circumferential or radial forces on theprimary vessel 702, thereby causing the primary vessel 702 to collapseinwardly against the float 704 Magnified view 902 shows the sealing ring500 tightened around the float and primary vessel system 700. Thesealing ring 500, having been placed at an interface of the buffy coat802 and the red blood cells 803, causes the primary vessel 702 tocollapse inwardly until a seal is formed between the primary vessel 702and the float 704. An outer wall of the sealing ring 500 may sit flushwith an outer wall of the primary vessel 702; the outer wall of thesealing ring 500 may extend past the outer wall of the primary vessel702; or, the outer wall of the primary vessel 702 may extend past theouter wall of the sealing ring 500. The sealing ring 500 remainstightened to maintain the seal, which prevents fluids from moving pastthe seal in any direction. The sealing ring 500 may also remain intension. Alternatively, the sealing ring 500 may be overtightened andthen the force applied to the sealing ring 500 is removed. The sealingring 500 may expand slightly, though still remains constricted.

To apply the sealing ring 500 and thereby form the seal, a clamp may beused to circumferentially apply a force directed toward the central axisof the primary vessel 702 to the sealing ring 500 and the float andprimary vessel system 700. The sealing ring 500 is placed around thefloat and primary vessel system 700 after the float and primary vesselsystem 700 have undergone density-based separation, such as bycentrifugation. The sealing ring 500 and float and primary vessel system700 are then placed into the clamp. The clamp may include a shelf tosupport the sealing ring 500 against the primary vessel 702. Operationof the clamp may be automated or may be performed manually.Alternatively, the clamp may form a seal between the float 704 andprimary vessel 702 without the inclusion of the sealing ring 500.Alternatively, a seal may be formed between the float 704 and theprimary vessel 702 such as by ultrasonic welding; or by applying heat ora temperature gradient to deform and/or melt the primary vessel 702 tothe float 704. For the sake of convenience, the methods are describedwith reference to the seal between the float and the primary vessel, butthe methods described below are not intended to be so limited in theirapplication and may be performed without the seal.

When operation of the clamp is automated, a motor causes translation ofeither a collet, including collet fingers, or a pressure member to causecompression of the collet fingers. The motor may be connected to thecollet or the pressure member by a shaft, such as a cam shaft, and oneor more gears. A base engages and holds the object. When the collet isdriven by the motor, the pressure member remains stationary. When thepressure member is driven by the motor, the collet remains stationary.The clamp may include a release, so as to cause the pressure member toslide off of the collet fingers 904, thereby removing the clampingforce.

Alternatively, the clamp may be, but is not limited to, a collet clamp,an O-ring, a pipe clamp, a hose clamp, a spring clamp, a strap clamp, ora tie, such as a zip tie. The clamp may be used without a sealing ringto provide a seal between a float and a tube.

The plasma 801 may be removed from the primary vessel 702, as shown inFIG. 10A, such as by pipetting, suctioning, pouring, or the like.Returning to FIG. 6, in block 608, a clearing fluid may be added to theprimary vessel along with a collector-canopy system. FIGS. 10B-10C showa clearing fluid 1002 having a density greater than at least the buffycoat 802 (i.e. may have a density greater than the buffy coat but lessthan the red blood cells, or may have a density greater than both thebuffy coat and the red blood cells, for example) being added to theprimary vessel 702. The collector-canopy system 400 is then added to theprimary vessel 702, as shown in FIG. 10D. The second end 208 of thecollector 200 forms a seal 1008 with the inner wall of the primaryvessel 702 to prevent fluid from flowing around the collector 200before, during, and after centrifugation. The seal 1008 may be formedbetween the second end 208 and an inner wall of the primary vessel tomaintain a fluid-tight sealing engagement before, during, and aftercentrifugation and to inhibit any portion of the suspension from beinglocated or flowing between an inner wall of the primary vessel and amain body 204 of the collector 200. The seal may be formed by aninterference fit, a grease (such as vacuum grease), an adhesive, anepoxy, thermal bonding, ultrasonic welding, clamping (such as with aring or clamp), an insert that fits between the second end 208 and theinner wall of the primary vessel, or the like. A lock ring 1004 may beplaced over the shoulder 216 of the collector 200 and the open end 710of the primary vessel 702 to inhibit translation of the collector 200relative to the primary vessel 702. When the collector-canopy system 400is inserted, a portion of the clearing fluid 1002 in the primary vessel702 may be discharged through the cannula 214 and stopped by the canopy402. The discharged fluid may flow out through the window 218 and intothe primary vessel 702, though remaining above the seal between thesecond end 208 and the inner wall of the primary vessel 702, as seen bythe dashed lines 406 in magnified view 1006 which is taken along theline VI-VI.

Returning to FIG. 6, in block 610, the canopy 402 may then be removedand a processing vessel 302 including the displacement fluid 312 may beinserted into the collector 200 to form the collector-processing vesselsystem 300, as seen in FIG. 10E. Magnified view 1010, which is across-section taken along the line VII-VII, shows the displacement fluid312 in the processing vessel 302 and the clearing fluid 1002 and thebuffy coat 802 in the primary vessel 702.

Returning to FIG. 6, in block 612, the system is then re-centrifuged.FIG. 10F shows the collector-processing vessel system 300 and theprimary vessel 702 undergoing centrifugation. Magnified view 1012, whichis a cross-section view taken along the line VIII-VIII, shows a snapshotof the exchange of fluids between the primary vessel 702 and theprocessing vessel 302. As the clearing fluid 1002, having a greaterdensity than the buffy coat 802, moves down in the primary vessel 702,the buffy coat 802 is cleared from the float 704. As the displacementfluid 312, having a density greater than the buffy coat 802 but lessthan the clearing fluid 1002, flows from the processing vessel 302 intothe primary vessel 702, the buffy coat 802 moves upwards within theprimary vessel 702 through the funnel 222 and the cannula 214, and intothe processing vessel 302. As shown in FIG. 10G, the buffy coat 802 isin the processing vessel 302, while the displacement fluid 312 and theclearing fluid 1002 are in the primary vessel 702.

The processing vessel 302 including the buffy coat 802 may then beremoved from the collector 200 to undergo further processing, analysis,storage, or the like. After removing the processing vessel 302, aprocessing solution may be added, though the processing solution mayhave already been in the processing vessel prior to retrieval of thetarget material. The processing vessel may be shaken, such as by avortex mixer. The processing solution (not shown), having been addedbefore shaking either in liquid form, in a dissolvable casing, or in abreakable casing, may then mix with the buffy coat to effect atransformation and form a buffy coat-processing solution mixture. Thebuffy coat-processing solution mixture may then be dispensed onto asubstrate, such as a microscope slide.

Alternatively, more than one displacement fluid may be used.Alternatively, more than one processing vessel may be used, such thateach processing vessel includes a different displacement fluid todisplace different fractions or materials of the suspension into therespective processing vessel. Consecutive fractions may be removed fromthe primary vessel by displacing the respective fractions with therespective displacement fluids. For example, a first processing vesselmay include a first displacement fluid to displace the plasma into thefirst processing vessel. A second processing vessel may include a seconddisplacement fluid to displace the buffy coat into the second processingvessel; the second processing vessel may also include the processingsolution to effect a change on the buffy coat.

The target material may be analyzed using any appropriate analysismethod or technique, though more specifically extracellular andintracellular analysis including intracellular protein labeling;chromogenic staining; nucleic acid analysis, including, but not limitedto, DNA arrays, expression arrays, protein arrays, and DNA hybridizationarrays; in situ hybridization (“ISH”—a tool for analyzing DNA and/orRNA, such as gene copy number changes); polymerase chain reaction(“PCR”); reverse transcription PCR; or branched DNA (“bDNA”—a tool foranalyzing DNA and/or RNA, such as mRNA expression levels) analysis.These techniques may require fixation, permeabilization, and isolationof the target material prior to analysis. Some of the intracellularproteins which may be labeled include, but are not limited to,cytokeratin (“CK”), actin, Arp2/3, coronin, dystrophin, FtsZ, myosin,spectrin, tubulin, collagen, cathepsin D, ALDH, PBGD, Akt1, Akt2, c-myc,caspases, survivin, pr^(kip), FOXC2, BRAF, Phospho-Akt1 and 2,Phospho-Erk1/2, Erk1/2, P38 MAPK, Vimentin, ER, PgR, PI3K, pFAK, KRAS,ALKH1, Twist1, Snail1, ZEB1, Fibronectin, Slug, Ki-67, M30, MAGEA3,phosphorylated receptor kinases, modified histones, chromatin-associatedproteins, and MAGE. To fix, permeabilize, or label, fixing agents (suchas formaldehyde, formalin, methanol, acetone, paraformaldehyde, orglutaraldehyde), detergents (such as saponin, polyoxyethylene,digitonin, octyl β-glucoside, octyl β-thioglucoside,1-S-octyl-β-D-thioglucopyranoside, polysorbate-20, CHAPS, CHAPSO,(1,1,3,3-Tetramethylbutyl)phenyl-polyethylene glycol or octylphenolethylene oxide), or labeling agents (such as fluorescently-labeledantibodies, enzyme-conjugated antibodies, Pap stain, Giemsa stain, orhematoxylin and eosin stain) may be used.

A solution containing a fluorescent probe may be used to label thetarget material, thereby providing a fluorescent signal foridentification and characterization. The solution containing thefluorescent probe may be added to the suspension before the suspensionis added to the vessel, after the suspension is added to the vessel butbefore centrifugation, or after the suspension has undergonecentrifugation. The fluorescent probe includes a fluorescent moleculebound to a ligand. The target material may have a number of differenttypes of surface markers. Each type of surface marker is a molecule,such an antigen, capable of attaching a particular ligand, such as anantibody. As a result, ligands can be used to classify the targetmaterial and determine the specific type of target materials present inthe suspension by conjugating ligands that attach to particular surfacemarkers with a particular fluorescent molecule. Examples of suitablefluorescent molecules include, but are not limited to, quantum dots;commercially available dyes, such as fluorescein, FITC (“fluoresceinisothiocyanate”), R-phycoerythrin (“PE”), Texas Red, allophycocyanin,Cy5, Cy7, cascade blue, DAPI (“4′,6-diamidino-2-phenylindole”) and TRITC(“tetramethylrhodamine isothiocyanate”); combinations of dyes, such asCY5PE, CY7APC, and CY7PE; and synthesized molecules, such asself-assembling nucleic acid structures. Many solutions may be used,such that each solution includes a different type of fluorescentmolecule bound to a different ligand.

The foregoing description, for purposes of explanation, used specificnomenclature to provide a thorough understanding of the disclosure.However, it will be apparent to one skilled in the art that the specificdetails are not required in order to practice the systems and methodsdescribed herein. The foregoing descriptions of specific embodiments arepresented by way of examples for purposes of illustration anddescription. They are not intended to be exhaustive of or to limit thisdisclosure to the precise forms described. Many modifications andvariations are possible in view of the above teachings. The embodimentsare shown and described in order to best explain the principles of thisdisclosure and practical applications, to thereby enable others skilledin the art to best utilize this disclosure and various embodiments withvarious modifications as are suited to the particular use contemplated.It is intended that the scope of this disclosure be defined by thefollowing claims and their equivalents.

We claim:
 1. A method for collecting a target material from asuspension, the method comprising the steps of: inserting a collectorinto an open end of a primary vessel that contains the suspension, thecollector comprising a cavity in a first end, an opening in a secondend, and a cannula to extend between the opening and the cavity;inserting a processing vessel into the cavity, the cannula to extendinto the processing vessel; adding a displacement fluid to theprocessing vessel, the displacement fluid having a density greater thanthe density of the target material; and centrifuging the primary vessel,the collector, the displacement fluid, and the processing vessel,wherein during centrifuging, the displacement fluid flows into theprimary vessel via the collector to displace the target material fromthe primary vessel, which flows into the processing vessel through thecollector via the cannula.
 2. The method of claim 1, further comprisingthe step of: separating the suspension into suspension fractions priorto inserting the collector into the primary vessel.
 3. The method ofclaim 2, further comprising the step of: adding at least one delineationfluid to the primary vessel before inserting the collector to providefurther separation between the target material and any non-targetmaterial.
 4. The system of claim 3, wherein the at least one delineationfluid has a density less than the non-target material and greater thanthe target material or a density that is greater than the non-targetmaterial and less than the target material.
 5. The method of claim 2,further comprising the step of: removing at least a portion of anon-target material from the primary vessel after the separating stepand before inserting the collector into the primary vessel.
 6. Themethod of claim 5, further comprising the step of: adding a clearingfluid to the primary vessel after the removing step, the clearing fluidhaving a density greater than at least the target material.
 7. Themethod of claim 6, further comprising the step of: adding a float to theprimary vessel prior to the separating step.
 8. The method of claim 7,further comprising the step of: forming a seal between the float and theprimary vessel by clamping after the separating step, the removing step,or adding the clearing fluid.
 9. The method of claim 1, wherein thecannula is a tube, a needle, or a non-coring needle.
 10. The method ofclaim 1, wherein the collector further comprises a shoulder extendingcircumferentially around a main body to inhibit further translation ofthe collector into the primary vessel.
 11. The method of claim 10,wherein the collector further comprises a lock ring to be placed overthe shoulder of the collector and the open end of the primary vessel toinhibit translation of the collector relative to the primary vessel. 12.The method of claim 1, wherein the collector further comprises anextension extending away from the first end and a lid to at leasttemporarily seal an opening within the extension, wherein the opening isin fluid communication with the cavity at the first end.
 13. The methodof claim 1, further comprising the step of: forming a seal between thesecond end of the collector and an inner wall of the primary vessel tomaintain a fluid-tight sealing engagement between the collector and theinner wall of the primary vessel.
 14. The method of claim 13, whereinthe seal between the second end of the collector and the inner wall ofthe primary vessel is formed by an interference fit, by a grease, byvacuum grease, by an adhesive, by an epoxy, by bonding, by thermalbonding, by welding, by ultrasonic welding, by clamping, by an insertthat fits between the second end and the inner wall of the primaryvessel, by an O-ring, or by a collar.
 15. The method of claim 13,wherein the collector further comprises a canopy in the cavity duringthe step of inserting the collector into the primary vessel, the canopyto inhibit any fluid that is discharged through the cannula fromescaping from the first end of the collector; and removing the canopybefore inserting the processing vessel into the collector.
 16. Themethod of claim 15, wherein the collector further comprises a windowwithin a main body to access at least a portion of the cavity.
 17. Themethod of claim 16, wherein the discharged fluid flows out through thewindow of the collector into the primary vessel and remains above theseal between the second end and the inner wall of the primary vessel.18. The method of claim 1, wherein the displacement fluid is selectedfrom the group consisting of: a solution of colloidal silica particlescoated with polyvinylpyrrolidone, a polysaccharide solution, iodixanol,an organic solvent, a liquid wax, an oil, a gas, olive oil, mineral oil,silicone oil, immersion oil, mineral oil, paraffin oil, silicon oil,fluorosilicone, perfluorodecalin, perfluoroperhydrophenanthrene,perfluorooctylbromide, organic solvents, 1,4-Dioxane, acetonitrile,ethyl acetate, tert-butanol, cyclohexanone, methylene chloride,tert-Amyl alcohol, tert-Butyl methyl ether, butyl acetate, hexanol,nitrobenzene, toluene, octanol, octane, propylene carbonate,tetramethylene sulfones, ionic liquids, a polymer-based solution, asurfactant, a perfluoroketone, perfluorocyclopentanone,perfluorocyclohexanone, a fluorinated ketone, a hydrofluoroether, ahydrofluorocarbon, a perfluorocarbon, a perfluoropolyether, silicon, asilicon-based liquid, phenylmethyl siloxane, and combinations thereof.19. The method of claim 1, wherein the processing vessel includes a plugin a closed end, the cannula to extend through the plug.
 20. The methodof claim 19, wherein the plug is composed of a resealable material.